Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.     

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79)

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.     

Conformations of peptides containing Z-alpha,beta-dehydroleucine (delta ZLeu). A comparison of Boc-Pro-delta ZLeu-Gly-NHEt and Boc-Pro-delta ZPhe-Gly-NHEt

Two tripeptides of the type Boc-Pro-delta ZX-Gly-NHEt (where X = Leu, Phe) have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r. and i.r. spectroscopy. These conformational studies indicated that delta ZLeu, similar to delta ZPhe, has a strong tendency to stabilize folded Type II beta-turn conformations when present at i + 2 position.     

Ultrasound induced damages and time bound recovery in mouse liver

Therapeutic ultrasound at 875 kHz at 10 and 15 W/cm2 intensity induced extensive damages in the liver of mouse. Total exposure of 5 min was spread over 5 days. Aqueous medium was avoided by coupling the transducer directly to the skin surface. Mild to extensive damages were noted. Complete distortion of hepatocellular architecture was noted in 15 W irradiated mice. However, there was almost complete recovery by 10th day following the last exposure.     

Regulation of seed number and female incitation of mate competition by a pH-dependent proteinaceous inhibitor of pollen grain germination in Leucaena leucocephala

Summary Leucaena leucocephala generally produces pods with more than 7–9 seeds. This is regulated by the stigmatic inhibition of pollen grain germination when the pollen grains are less than a critical number in the stigma. This number-dependent inhibition of pollen grain germination is effected by a pH-dependent proteinaceous inhibitor active at the stigmatic pH. Only when the pollen grains in the stigma exceed the critical number, they inactivate the inhibitor by collectively raising the stigmatic pH and thus overcoming the inhibition. The adaptive significance of such pre-fertilization mechanism for the female in inciting mate competition among the pollen grains is discussed. The evolution of en masse pollen grain dispersal units is explained as a sexual selection strategy by males in response to such stigmatic inhibition by females.     

Seed abortion in wind-dispersed pods of Dalbergia sissoo: maternal regulation or sibling rivalry?

Summary Dalbergia sissoo, a wind-dispersed tropical tree, shows a positively skewed distribution of seeds per pod. This is attributed to the enhanced dispersal advantage of few-seeded pods due to their reduced wing loading (ratio of weight to pod surface area) and low settling velocity. The proximate mechanisms causing the positively skewed distribution were investigated. The distribution could not be attributed to the distribution pattern of ovule number per ovary, pollen grain limitation, lack of ovule fertilization, or post-fertilization elimination of many-seeded pods. Rather, it was caused by the post-fertilization abortion of seeds within a pod 2 weeks after fertilization. This intra-pod seed abortion (IPSA) is due to a dominance hierarchy of fertilized ovules from the distal (near stigma) to the basal end, generated by the temporal differences in fertilization. The dominant developing seeds at the distal end cause the abortion of others through the production and diffusion of an aborting agent. When the dominance hierarchy of the siblings is not intense, pods are formed with more than one seed. We argue that the positively skewed distribution of seeds per pod is not due to maternal regulation but is a result of sibling rivalry. We propose that this sibling rivalry is generated by genetic differences in pollen grain fitness and disucss the results in the context of parent-offspring conflict.     

Association of poly(A) polymerase with U1 RNA

Previous studies (Stetler, D. A., and Jacob, S. T. (1984) J. Biol. Chem. 259, 7239-7244) have shown that poly(A) polymerase from adult rat liver (liver-type) is structurally and immunologically distinct from the corresponding rat hepatoma (tumor-type) enzyme. When hepatoma 7777 (McA-RH 7777) cells were labeled with [32P]inorganic phosphate, followed by immunoprecipitation with anti-hepatoma poly(A) polymerase antibodies and analysis of the RNAs in the immunoprecipitate, only one labeled small nuclear RNA corresponding to U1 RNA was found. Preimmune sera did not form a complex with U1 RNA. Hepatoma poly(A) polymerase antisera did not immunoprecipitate U1 RNA or any other small nuclear RNA from a cell line (H4-11-EC3) which does not contain the tumor-type poly(A) polymerase. Immunoblot analysis of hepatoma 7777 nuclear extract or purified poly(A) polymerase with anti-ribonucleoprotein antisera did not show any cross-reactivity of the latter sera with poly(A) polymerase. The major RNA immunoprecipitated from the hepatoma nuclear extracts using trimethyl cap (m3G) antisera corresponded to the RNA immunoprecipitated with poly(A) polymerase antisera. These data indicate that U1 RNA is closely associated with poly(A) polymerase and suggest the potential involvement of this RNA in the cleavage/polyadenylation of mRNA precursor.